Summary: Recent studies from this laboratory using the neuronal cell line H32, which expresses endogenous V1 VP receptors, showed that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. These actions of VP are largely mediated by mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), but also involves other signaling pathways. Studies during the past year have examined the role of protein kinases C (PKC) and B (PKB) mediating VP induced antiapoptosis in H32 cells. Serum deprivation increased PKC delta, but not PKC alpha;or PKC beta;activity, while VP increased PKC alpha and PKC beta without affecting PKC delta activity. Inhibition of PKC delta prevented caspase-3 activation, indicating that this PKC subtype mediates the proapoptotic actions of serum deprivation. Simultaneous inhibition of PKC alpha and beta, and MAP kinase abolished VP-induced Bad phosphorylation, but it only partially prevented caspase-3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of PI3K/protein kinase B (Akt). The data demonstrate that VP exerts antiapoptosis through multiple pathways;while PKC alpha and beta together with ERK/MAP kinase activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway. The above mentioned studies in H32 cells demonstrate that VP has antiapoptotic actions in a neuronal cell line and suggest that VP has neuroprotective actions. To test this hypothesis and determine whether VP is antiapoptotic in hippocampal neurones, primary cultures of these neurones were used to examine the effect of VP on neuronal culture supplement (B27) deprivation-, or glutamate-induced apoptosis, and the signaling pathways mediating the effects. Removal of B27 supplement from the culture medium for 24 hours or addition of glutamate (3 to 10 micromolar) significantly decreased neuronal viability and increased Tdt-mediated dUTP nick-end labeling (TUNEL) staining and caspase-3 activity. These changes are consistent with apoptotic cell death, and were significantly reduced by addition of VP (10nM), indicating that the peptide has antiapoptotic actions in the primary neuronal cultures. These antiapoptotic effects of VP were completely blocked by a V1 but not a V2 receptor antagonist, indicating that they are mediated via V1 VP receptors. The signaling pathways mediating the antiapoptotic effect of VP in neurones involves activation of MAPK/ERK and IP3/Akt, similar to the observations reported in the cell line H32. This was shown by the transient increases in phospho-ERK and phospho-Akt after incubation with VP revealed by western blot analyses, and the ability of specific inhibitors to reduce the inhibitory effect of VP on caspase-3 activity and TUNEL staining by 70% and 35%, respectively. These studies demonstrate that VP has antiapoptotic actions in hippocampal neurones, an effect which is mediated by the MAPK/ERK and PI3/Akt signaling pathways. The ability of VP to reduce nutrient deprivation or glutamate overstimulation-induced neuronal death suggests that VP acts as a neuroprotective agent within the brain. Since VP is released in the hippocampus during stress, the peptide may contribute to preventing damaging effects of stress in neurones.